Immunotherapy is a promising approach for treating patients with advanced breast cancer. However, immunosuppressive microenvironments induced by regulatory T cells (Treg) present a major barrier to successful anti-tumor immunotherapy. A better understanding of the suppressive mechanisms utilized by different types of tumor-infiltrating Treg cells is essential for the development of novel strategies to trea human cancers. Conventional Treg cells have been extensively studied, however, little is known about the negative regulation mediated by ?? T cells in anti-tumor immune responses in cancer patients. We recently discovered high percentages of ?? Treg cells existing among the tumor-infiltrating lymphocytes (TILs) of breast tumor patients, which are strongly negatively correlated with clinical outcomes. We further identified a novel suppressive mechanism whereby ?? Treg cells induce senescence in T cells and dendritic cells (DCs) that then also develop potent suppressive activity. Therefore, it is critical to further identify the molecular mechanisms responsible for ?? Treg-induced senescence and suppressive effects, and to develop strategies to reverse senescence induction mediated by ?? Treg cells. Our long-term goals are to identify the immunoregulatory mechanisms utilized by tumor-associated Treg cells and develop strategies to modify their suppressive effects for improved cancer treatment. The central hypotheses of this proposal are that: 1) breast tumor-derived ?? Treg cells not only can directly suppress nave/effector T cells and DCs, but also can direct their differentiation into senescent cells with altered biological functions that amplify immune suppression; 2) immune suppression and senescence induction mediated by ?? Treg cells can be blocked by TLR8 signaling, thereby resulting in enhanced anti-tumor immunity. Specific Aim 1 seeks to determine the molecular and biological alterations induced by human breast tumor-derived ?? Treg cells in nave /effector T cells leading to T cell senescence. We will then identify the importance of ERK1/2 and p38 signaling in controlling senescence induction in ?? Treg-treated responder T cells. Aim 2 will investigate the amplified immune suppression in adaptive immunity mediated by ?? Treg-induced senescent DCs and further identify the mechanisms responsible for the tolerogenic functions of senescent DCs. We will then perform in vivo studies to investigate how ?? Treg- induced senescent DCs affect tumor-specific effective immune responses in breast cancer models. Aim 3 will first dissect the mechanisms responsible for TLR8 signaling-mediated reversal of ?? Treg suppression and senescence induction in responder T cells involving glucose metabolism changes in ?? Treg cells. We will then investigate the enhancement of anti-tumor immunity through manipulation of TLR8 signaling and/or glucose metabolism in ?? Treg cells preventing generation of senescent tumor-specific T cells in vivo in breast cancer immunotherapy models. A positive outcome from these studies should lead to novel strategies for manipulation of ?? Treg-induced suppression for the treatment of human breast cancer and other cancers as well.